Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag.
Like other members of the transforming growth factor beta (TGF-beta) family of growth factors, the biological activity of TGF-beta2 is believed to be regulated by the formation and dissociation of multiprotein complexes. To isolate the molecular complex formed by TGF-beta2 secreted by hypertrophic chondrocytes we have used expression of TGF-beta2 fused with the humanized, tandem affinity purification (hTAP) tag and mass spectrometry for the identification of interacting proteins. The hTAP synthetic gene was assembled by systematically replacing the rare codons of the original TAP tag with codons most preferred in highly expressed human genes to circumvent the poor translation efficiency of the original TAP tag in animal cells. TGF-beta2 was shown to interact with Type X collagen and this interaction confirmed using V5 tagged TGF-beta2. Functional interaction was suggested by the inhibition of TGF-beta2 activity by type X collagen in culture and the influence of a mutation in type X collagen on the distribution of TGF-beta2 in growth cartilage.
Yang
Maozhou
M
Bone and Joint Center, Henry Ford Hospital, 2799 West Grand Blvd., MI 48202, USA.
Wang
Xinli
X
Zhang
Liang
L
Yu
Chiyang
C
Zhang
Bingbing
B
Cole
William
W
Cavey
Greg
G
Davidson
Paula
P
Gibson
Gary
G
D000293:Adolescent; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002645:Chickens; D002648:Child; D019902:Chondrocytes / Q000378:metabolism; D003001:Cloning, Molecular; D024121:Collagen Type X / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D009154:Mutation; D009842:Oligopeptides; D010009:Osteochondrodysplasias / Q000473:pathology; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014162:Transfection; D053781:Transforming Growth Factor beta2 / Q000235:genetics / Q000378:metabolism*
D000345:Affinity Labels; D024121:Collagen Type X; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D053781:Transforming Growth Factor beta2; C085706:FLAG peptide